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Biomedical subjects

P L Dutton

Publications and source records attributed to P L Dutton.

At least 91 records · Page 5Linked to original sources
PubMed1982

Reduction of cytochromes b6 and f in isolated plastoquinol-plastocyanin oxidoreductase driven by photochemical reaction centers from Rhodopseudomonas sphaeroides.

Photochemical reaction centers isolated from the bacterium Rhodopseudomonas sphaeroides are able to donate electrons to cytochromes b6 and f in the plastoquinol,-platocyanin oxidoreductase isolated from spinach chloroplasts. The reduction reactions occur only after the second single turnover flash, in a reaction which is sensitive to inhibitors of the reactions in the chloroplast membranes. When all the components of the b6f complex are oxidized prior to activation, both cytochromes b6 and f are reduced after the second flash. If cytochrome f is reduced prior to activation, cytochrome b6 is still reduced after the second flash, but as the potential is lowered so that the Rieske iron-sulfur cluster is reduced prior to activation, the reduction of cytochrome b6 fails. The b6f complex thus functions in such a way that a two-electron redox couple, probably a quinone, is capable of reducing both cytochrome b6 and cytochrome f, the latter via the Rieske iron-sulfur cluster, in a coupled reaction where both electrons must leave the two-electron carrier. Cytochrome b6 is thus reduced in a manner analogous to "oxidant-induced reduction."

Bacterial Proteins↗
PubMed1982

The location of redox centers in biological membranes determined by resonance x-ray diffraction. I. Observation of the resonance effect.

We have developed resonance X-ray diffraction methods to locate for the first time intrinsic metal atoms associated with redox centers within biological membrane systems. The study of membranes containing dilute concentrations of resonant scatterers has been made possible by the development of synchrotron radiation sources of X-rays. The technique permits altering the scattering power of a particular atom relative to others by varying the incident X-ray energy. Thus, this method may be used to locate a metal atom within a complex integral protein without chemical modification of the membrane. We present resonance diffraction data taken with synchroton radiation for two different membrane systems: cytochrome oxidase incorporated into lipid vesicles and a photosynthetic reaction center-cytochrome c complex also reincorporated into lipid vesicles.

Cell Membrane↗
PubMed1982

The location of redox centers in biological membranes determined by resonance x-ray diffraction. II. Analysis of the resonance diffraction data.

In the preceding paper (Stamatoff, J., Eisenberger, P., Blasie, J.K., Pachence, J.M., Tavormina, A., Erecinska, M., Dutton P.L. and Brown, G. (1982) Biochim. Biophys. Acta 679, 177-187), we described the observation of resonance X-ray scattering effects from intrinsic metal atoms associated with redox centers in membrane proteins on the lamellar X-ray diffraction from oriented multilayers of reconstituted membranes. In this paper, we discuss the possible methods of analysis of such data and present the results of our model refinement analysis concerning (a) the location of the cytochrome c heme iron atom in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex and (b) the location of the heme a and a3 iron atoms in the profile structure of a reconstituted membrane containing cytochrome oxidase. The former results are of special importance because they provide a test of the validity of the resonance diffraction data and the methods of analysis, since the location of cytochrome c in the reaction center-cytochrome c membrane profile is known independently of the resonance diffraction experiments.

Animals↗
PubMed1982

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers. Response under conditions of single electron turnover.

Light-induced electric current and potential responses have been measured across planar phospholipid membranes containing reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides. Under conditions in which the reaction centers are restricted to a single electron turnover, the responses can be correlated with the light-induced electron transfer reactions associated with the reaction center. The results indicate that electron transfer from the bacteriochlorophyll dimer to the primary ubiquinone molecule, and from ferrocytochrome c to the oxidized dimer occur in series across the planar membrane. Electron transfer from the primary to secondary ubiquinone molecule is not electrogenic.

Bacteriochlorophylls↗
PubMed1981

The reaction center profile structure derived from neutron diffraction.

Both reaction center protein from the photosynthetic bacteria Rhodopseudomonas sphaeroides and egg phosphatidylcholine can be deuterium labelled; the reaction center protein can be incorporated into the phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. The lipid profile and the reaction center profile within these reconstituted membrane profiles were directly determined to 32 A resolution using lamellar neutron diffraction from oriented membrane multilayers containing either deuterated or protonated reaction centers, and either deuterated or protonated phosphatidylcholine. The 32 A resolution reaction center profile shows that the protein spans the membranes, and has an asymmetric mass distribution along the perpendicular to the membrane plane. These results were combined with previously described X-ray diffraction results in order to extend the resolution of the derived reaction center profile to 9 A.

Bacterial Proteins↗
PubMed1981

The thermodynamic properties of some commonly used oxidation-reduction mediators, inhibitors and dyes, as determined by polarography.

The oxidation-reduction midpoint potentials (Em) of the following compounds have been measured in the range of pH from 3 to 12 by polarography: methyl viologen; benzyl viologen; 2-hydroxy-1,4-naphthoquinone; 2-hydroxy-1,4-anthraquinone; N,N,N',N',-tetramethyl-p-phenylenediamine; 2,3,5,6-tetramethyl-p-phenylenediamine; phenazine; N-methylphenazonium methosulfate; N-methylphenazonium sulfonate methosulfate; N-ethylphenazonium ethosulfate; pyocyanine; neutral red; safranin; phenol red; chlorophenol red; cresol red; bromocresol purple; 2,5-dibromo-3-methyl-6-isopropylbenzoquinone and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Many of these previously assumed to have a simple behavior in this range have proven to be rather more complicated, and several anomalous observations have been reconciled.

Dibromothymoquinone↗
PubMed1981

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas sphaeroides.

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c2 oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of antimycin, after subtraction of contributions due to absorption changes from cytochrome c2, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll. 2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift. 3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reductions 3- to 4-fold under certain if not all conditions. 4. A maximum of approximately 0.6 cytochrome b-560 (Em(7) = 50 mV, n = 1, previously cytochrome b50) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c2 oxidoreductase. 5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer. 6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c2 oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Qz) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c2 oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Qz, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c2 oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (QII) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Qz, respectively.

Bacteriochlorophylls↗
PubMed1980

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides.

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c2, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation (t 1/2 3-5 microseconds). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (102 ms) antimycin-sensitive phase of ferricytochrome c2 reduction, attributed to a specific ubiquinone-10 molecule (Qz), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Qz (QzH2) reduces the Rieske Center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c2 reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center. UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from gy 1.90 to gy 1.89, and shifts the Em,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c2, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of HII(+); these may reflect additional sites of action of the inhibitor.

Antimycin A↗
PubMed1980

Construction of a flash-activated cyclic electron transport system by using bacterial reaction centers and the ubiquinone-cytochrome b-c1/c segment of mitochondria.

Single-turnover electron transfer within the mitochondrial complex III has been studied by combining, in solution, the isolated complex from bovine heart with detergent-solubilized reaction centers of Rhodopseudomonas sphaeroides. Initiation of electron transfer by short flash activation resulted in the prompt oxidation of cytochrome c and reduction of cytochrome b. The subsequent reduction of ferricytochrome c was observed to be concomitant with the oxidation of the ferrocytochrome b, both reactions being inhibited by the addition of actimycin A. The rate of electron transfer through complex III is dependent upon the ambient redox potential poise in a way that is consistent with the presence of a redox component, presumably analogous to the photosynthetic ubiquinone Qz, which is an obligatory intermediate in electron transfer between cytochromes b and c. These results demonstrate cyclic electron transfer in a constructed assembly of mitochondrial complex III, cytochrome c, and photochemical reaction centers.

Animals↗
PubMed1979

The recognition of a special ubiquinone functionally central in the ubiquinone-cytochrome b-c2 oxidoreductase.

Although the energy conserving membranes of the photosynthetic bacterium Rhodopseudomonas sphaeroides contain a 25 (+/- 3)-fold molar excess of ubiquinone over the photochemical reaction center, the activity of the ubiquinone-cytochrome b-c2 oxidoreductase is unaffected by quinone extraction until only 3, or at most 4, ubiquinones remain; only then does further extraction prevent the function of the oxidoreductase. Since 2 of these last ubiquinones are integral parts of the photochemical reaction center, we conclude that the ubiquinone-cytochrome b-c2 oxidoreductase requires only 1, or at most 2, molecules of ubiquinone-10 for its function. Earlier kinetic data identified a major electron donor to ferricytochrome c2 as a single molecule (known as Z) which requires 2 electrons and 2 protons for its equilibrium reduction. Hence, we identify a single molecule of quinone, probably ubiquinone-10 in a special environment, as a major electron donor to ferricytochrome c2 in the ubiquinone cytochrome b-c2 oxidoreductase.

Cytochrome c Group↗
PubMed1979

Structural studies on reconstituted reaction center-phosphatidylcholine membranes.

Reaction center protein, isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26 mutant, was incorporated into phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. Cytochrome c, added to preformed reaction center-phosphatidylcholine vesicles, rapidly reduced up to 90% of the laser-generated (BChl)2+ of the reaction center (with kinetics of electron transfer similar to those in the chromatophore membrane) which suggests that the portion of the reaction center which accommodates functional cytochrome c binding sites is exposed predominantly on the exterior of the vesicles. Unit cell electron density profiles were derived from lamellar X-ray diffraction from oriented reaction center-phosphatidylcholine membrane multilayers at varying lipid/protein ratios. The analysis of these profiles showed that the reaction center protein incorporates into the phosphatidylcholine membrane with unique sidedness and that the profile of the reaction center protein itself is asymmetric and spans the membrane.

Bacterial Proteins↗
PubMed1979

Electron and proton transport in the ubiquinone cytochrome b-c2 oxidoreductase of Rhodopseudomonas sphaeroides. Patterns of binding and inhibition by antimycin.

The effect of antimycin on the ubiquinone cytochrome b-c2 (Q b-c2) oxidoreductase of the photosynthetic bacterium Rhodopseudomonas sphaeroides has been studied under controlled oxidation-reduction potential (Eh) conditions by equilibrium measurements and by rapid kinetic analysis of single turnover flash.induced electron and proton translocations. 1. Antimycin shifts the alpha-band of ferro b50 (lambda max 560 nm) by 1 to 2 nm toward the red but has no apparent effect on the equilibrium oxidation-reduction midpoint potential of the cytochrome. 2. This red shift is proportional to the antimycin added until a "titer" of 0.7 +/- 0.1 antimycin per reaction center (RC) is approached. With a similar titer antimycin essentially abolishes the following millisecond reactions activated by saturating single turnover flashes: reduction of ferri c2, oxidation of ferro b, Phase III of the membrane-potential-indicating band shift of endogenous carotenoid pigments, and the uptake of 1 of the 2 protons taken up per electron transferred. Such titrations indicate that the binding (KD approximately 10(-9) m) and mode of inhibition of antimycin are noncooperative and are independent of the membrane's coupling status and of the pH and Eb over the range in which electron transport is operative. 3. In the presence of excess antimycin a partial recovery of ferri c2 reduction is seen when the intensity of the flash is diminished, but only at Eh values such that Z (a special quinone serving as reductant for ferri c2) is reduced but b50 is oxidized before activation. These results are consistent with the following model. Each Q b-c2 oxidoreductase complex includes one antimycin binding site, one b50, and one Z. These complexes and the c2 . RC complexes, present in an 0.7:1 ratio, are to some degree mobile with respect to each other. Ferri b50 can be reduced either via the quinones of the RC or via Z in a reaction also involving c2. The former route is kinetically dominant in the presence of antimycin, but the latter route is the means for "oxidant-induced reduction" and depends on the collisional interaction of the oxidoreductase and c2 . RC complexes. Antimycin interferes with neither of these two routes but does inhibit the oxidation of ferro b50; all the other inhibitory effects are consequent on this.

Antimycin A↗
PubMed1979

Ubiquinone in Rhodopseudomonas sphaeroides. Some thermodynamic properties.

In Rhodopseudomonas sphaeroides chromatophores there are 25 +/- 3 ubiquinone (Q) molecules/reaction center protein. They comprise several thermodynamically and functionally different ubiquinone complements. There are approx. 19 ubiquinones (Em7 = 90 mV) in the main ubiquinone complement which, within experimental resolution, appears thermodynamically homogenous and follows the redox reaction Q + 2e + 2H+ in equilibrium with QH2 from pH 5--9. A method which takes advantage of the 2H+ bound/molecule of Q reduced is described for measuring the time course of light-activated reaction center-driven reduction and oxidation of the 19 Q complement. No stable semiquinones were detected in the constitutents of the 19 Q complement. There are approx. 6 ubiquinones of lower Em which are currently unaccounted for, although one or possibly two of these can be assigned to the quinones of the reaction center protein. The remainder may be associated with the NADH-ubiquinone oxidoreductase.

Aerobiosis↗
PubMed1979

Factors controlling the binding of two protons per electron transferred through the ubiquinone and cytochrome b/c2 segment of Rhodopseudomonas sphaeroides chromatophores.

1. On every turnover, 2.0 protons can be bound by the membrane for each single electron moving through the Q-b/c2 oxidoreductase. 2. One proton (H+II) binding reaction is, and one (H+I) is not, sensitive to antimycin. 3. The redox states of electron transfer components other than the proton binding agents can affect both the rate of proton uptake and the apparent pK values on the agents binding the protons. 4. The presence of valinomycin under certain well-defined conditions can strongly influence the value of the measured pK on the H+II binding agent.

Bacterial Chromatophores↗
PubMed1979

Electrogenic events in the ubiquinone-cytochrome b/c2 oxidoreductase of Rhodopseudomonas sphaeroides.

The reductant of ferricytochrome c2 in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c2 by ZH2 is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c2 reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH2 and ferricytochrome c2, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c2 reduction at pH 7.0 and at 24 degrees C was 2 - 10(9) M-1 - s-1, but this varied with pH, being 5.1 - 10(8) M-1 = s-1 at pH 5.2 and 4.3 - 10(9) M-1 - s-1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.

Bacterial Chromatophores↗